1. Field of the Invention
The present invention relates to a open-pore foamed cellulose material used as carriers for ion exchangers and culturing animal cells, which is easy to handle and has a large surface area per unit weight.
More particularly, this invention concerns a cellulose ion exchanger obtained by the introduction of an ion-exchangeable functional group in an open-pore foamed cellulose material, which is easier to handle and larger in terms of the surface area per unit weight than conventional gel-like ion exchangers. This invention is also directed to an animal cell culture carrier which is easier to handle than conventional animal cell culture microcarriers and can culture animal cells while the cells are immobilized thereon at high densities, without deteriment to an environment well suited for cell growth.
2. Statement of the Prior Art
Industrial ion exchangers so far used in the art has their origin in a discovery by Adam et al in England in 1935 that condensates of phenolic compounds with formalin absorb alkalis while condensates of aniline-based compounds with formalin do acids. The commercialization began with success for the first time in Germany in 1938. Since then, they have enjoyed steadily increases indispensable to industrial purposes, inter alia, water disposal.
Most of these ion exchangers, based on resins, are of a hydrophobic nature on their surfaces. Depending upon what is ion-exchanged or, in more illustrative terms, when polymeric materials, e.g. hydrophilic biopolymers such as proteins, are ion-exchanged, they may often produce unfavourable effects due to their hydrophobic interaction with such polymers. This is for one thing, attributable to difficulty involved in elution operation, partly because ion bonds take no part in adsorption; for another, repellency takes place through electrostatic or van der Waals' force, resulting in a serious drop in adsorptivity.
Cellulose-based ion exchangers developed by Sober et al in 1954, on the other hand, are of so strong a hydrophilic nature on their surfaces, that if they are used in aqueous media, the surfaces of their supports can be taken as behaving just like an aqueous solution. To put it another way, neither adsorption nor repellency takes place through electrostatic force other than their ionic interaction with what is ion-exchanged. Therefore, such ion-exchangers have a great advantage of being able to control in an easy operation the adsorption and elution of matter, even though it is a sterically sophisticated biopolymer such as protein or lipid, in which one molecule includes hydrophilic and hydrophobic moieties with the electrostatic force therebetween being well balanced in an aqueous system. This is because no interaction but three types of electrostatic force takes place between three sets of polymer and water; polymer and functional groups the ion exchangers have; and functional groups the ion exchangers have and water.
Such ion exchangers based on cellulose supports are now commercialized in sheet (planar and filter sheets), powdery, granular and gel-like forms.
When carrying out cell culture, most of animal cells cannot proliferate without deposition onto the surface of solid matter. For that reason, some solid surface should be in store, whatever the culture scale is. In an effort to achieve the mass culture of cells in particular, every possible means is now used to increase the surface area per culture volume, thereby increasing productivity. Of these means, the most standard of all is a microcarrier culture technique, according to which microcarriers, each having a relatively large surface area, are suspended in a culture medium to increase the culture area available per culture volume to a maximum. The microcarriers, designed to culture animal cells, are obtained by forming polymers such as polysaccharides or polystyrene into beads having a particle size of 100 to 300 .mu.m, on which the cells are to be immobilized. For promoting deposition of cells onto the microcarriers, they may be either coated thereon with collagen or positively charged by chemical synthesis. The former collagen type of carrier has an advantage of showing higher bioaffinity than does naturally occurring collagen, whereas the latter charge type of carrier has several advantages of being able to immobilize floating cells which cannot otherwise be fixed in place and capable of being repeatedly used after the removal of cells using such enzymes as a protease etc. Thus, both types of carriers may be selectively used depending upon what they are used for. The charge type of carrier is prepared by the introduction of ternary amino groups represented by a diethylaminoethyl group or quaternary amino groups as cations, typically available in the trade names of Cytodex 1 and Cytodex 2 made by Pharmacia Co., Ltd.
Having one limited use in view of morphology, currently available cellulose ion exchangers may be used on laboratorial scales but have a disadvantage of being often unsuited for use at an industrially extensive scale.
In brief, sheet-form ion exchangers are used in such a way that paper filters are used. For instance, they have been primarily employed as the immobilizing layers for paper chromatography. They may also be used possibly in another way; they may be packed in columns through fluids flow for ion exchange, wherein an ultrafiltration membrane would be spirally found as the immobilzing film for the so-called column chromatography. However, experience teaches that they cannot be used for that purpose, since there is possibility that the film may break for lack of strength and by the force generated by the flowing of fluids.
Even when they are applied to filtration according to the average procedure of using paper filters, it is impossible to make the filtration area larger than the required space, if they are used in a planar form, thus failing to increase the ion exchange capacity. For this reason, they cannot be used without subjecting them to special morphological treatments.
Powdery ion exchangers may possibly be used as the fixed layer for thin-layer chromatography. However, such use has been rarely made for the fixed layer for column chromatography, in which they are packed for the ion exchange of a sample fluid, because the size of the particles renders it impossible to increase the flow rate of the sample fluid and to enhance the ion exchange efficiency.
The powdery ion exchangers are also ill suited for use with the so-called batchwise systems wherein they are put directly into a sample fluid to be ion exchanged, thereby selectively adsorbing the required substances from the sample fluid. This is because not only is the recovery of the necessary substances timeconsuming but their washing takes too much time as well.
Thus, these two types of ion exchangers may have found their use for analytical purposes alone, as there is clear-cut evidence that they are not well fit for use at an extensive scales.
On the other hand, granular or gel-form ion exchangers have been primarily used as chromatographic fixed layers in which they are packed, or for selectively adsorbing the required substances from a sample fluid in which they are put. However, their use is limited to narrow conditions (to such low flow rates so as not to produce pressure in a state of low ionic strength) because of the following demerits. For one thing, they are crushed flat due to their low physical strength when they receive the pressure or shear force generated by increased flow rates or stirring; for another, they are likely to contract or change in diameter when sample fluids of high ionic strength are used with them, giving rise to changes in their packing in columns and to channeling.
From the practical standpoint, limited use is not desirable, since it makes little contribution to the advancement of technology. Especically when a certain type of technology has limitations to scaling up, there is only limited use, e.g. analytical use, making poor its future industrial prospects.
Turning now to animal cell culture carriers, the most popular standard ones are a microcarrier having such a large surface area as to mass culture cells. However, that microcarrier is still less satisfactory in terms of the surface area. Thus, there is a strong demand among various animal cell-related industries for the development of a carrier enabling cell culture to be carried out at much higher densities. Moreover, when microcarriers are used with turbulently flowing culture solutions or excessively packed in culture solutions, there is an increased chance of their colliding with one another with large impact, so that the immobilized cells suffer damage to such an extent that they cannot survive. In the case of microcarriers, these problems are attributable to the fact that it is only their surfaces, not at some place in them, where cell immobilization takes place.
Taken altogether, we have reached the conclusion that the basic defects of cellulose ion exchangers and animal cell culture carriers can all stem from their morphological construction.